Nucleoside-5'-diphosphates containing purine or pyrimidine heterocycles are useful intermediates in the synthesis of biologically active compounds such as oligoribonucleotides. Nucleoside-5'-diphosphates are sources of energy and phosphoric acid in cellular metabolic processes, and are important in the biosynthesis of nucleic acids.
Ion exchange chromatography is commonly used to purify a variety of materials, including pharmaceuticals, on a manufacturing scale. There is a wide amount of literature available on various processes (Duolite.RTM. Ion-Exchange Manual, Diamond Shamrock Chemical Company, 1969).
Nucleoside-5'-diphosphates are commonly synthesized by reacting an activated 5'-nucleotide, such as a nucleoside-5'-monophosphoramidate or 1-diphenyl-2-(5'-nucleoside)diphosphate, with multiple equivalents of a tertiary amine salt of phosphoric acid in an anhydrous organic solvent (Michelson, Biochem. Biophys. Acta (1964) 91:1-13; Moffat and Khorana, J. Am. Chem. Soc. (1961) 83:649-658; U.S. Pat. No. 3,534,017 (1970)). Formation of by-products during the synthesis such as 1,2-di(5'-nucleoside)diphosphate (NPPN) and 1,3-di(5'-nucleoside)triphosphate (NPPPN) usually occur from the reaction of the activated 5'-nucleotide with 5'-nucleotide and nucleoside-5'-diphosphate, respectively.
NDPs are usually purified directly from the reaction media by ion exchange chromatography. The reaction masses are sometimes treated with activated carbon to remove organic solvents and inorganic phosphate before ion exchange chromatography. In other methods, organic solvents are removed by vacuum distillation and the residue reconstituted with water before ion exchange purification. For large-scale NDP manufacturing, it is desirable to purify NDPs directly from the reaction mass by ion exchange chromatography without the additional carbon treatment or vacuum distillation.
The ion exchange purification schemes most often used involve absorbing the nucleoside-5'-phosphate derivatives onto a column of strongly basic ion exchange resin containing quaternary polymer-bound trimethylamine sites (Type 1 resins) or quaternary polymer-bound dimethylmonoethanolamine sites (Type 2 resins) in the chloride form. The compounds are then individually eluted with dilute acid or dilute acid and salt solutions. The purification of uridine-5'-diphosphate and uridine-5'-triphosphate by such a method is described by Fujimoto and Teranishi in U.S. Pat. No. 3,509,128 (1970).
We have found that ion exchange systems similar to these were unable to separate certain nucleoside-5'-diphosphates, such as those containing purine or pyrimidine heterocycles without a primary amine constituent, from other phosphorylated impurities present in the chemical reaction matrix, such as NPPN. In all of the ion exchange chromatography systems we tried using acid and/or alkali metal salt solutions, NPPN co-eluted with NDP.
Experiments to test relative absorption of these compounds on the strongly basic ion exchange resin showed that NPPN was selectively retained over NDP by the strongly basic ion exchange resin in basic solutions, but not selectively retained in acidic solutions. Therefore, it appeared advantageous to develop a method to elute IDP under basic conditions.
Strongly basic ion exchange resins have a low affinity for the hydroxide anion; therefore, most ion exchange purifications using strongly basic ion exchange resins are performed using acidic eluents. Although hydroxide ion will exchange with chloride ion on strongly basic ion exchange resin, hydroxide ion, even in high concentration, is not strong enough to exchange with nucleoside-5'-phosphate derivatives on the resin. For this reason, the hydroxide ion has not been used in ion exchange chromatography purifications of phosphate derivatives using strongly basic resins.